Post-reperfusion acute MR diffusion in stroke is a potential predictor for clinical outcome in rats.

Middle cerebral artery occlusion (MCAO) models show substantial variability in outcome, introducing uncertainties in the evaluation of treatment effects. Early outcome predictors would be essential for prognostic purposes and variability control. We aimed to compare apparent diffusion coefficient (ADC) MRI data obtained during MCAO and shortly after reperfusion for their potentials in acute-phase outcome prediction. Fifty-nine male rats underwent a 45-min MCAO. Outcome was defined in three ways: 21-day survival; 24 h midline-shift and neurological scores. Animals were divided into two groups: rats surviving 21 days after MCAO (survival group, n = 46) and rats dying prematurely (non-survival/NS group, n = 13). At reperfusion, NS group showed considerably larger lesion volume and lower mean ADC of the initial lesion site (p < 0.0001), while during occlusion there were no significant group differences. At reperfusion, each survival animal showed decreased lesion volume and increased mean ADC of the initial lesion site compared to those during occlusion (p < 10-6), while NS group showed a mixed pattern. At reperfusion, lesion volume and mean ADC of the initial lesion site were significantly associated with 24 h midline-shift and neurological scores. Diffusion MRI performed soon after reperfusion has a great impact in early-phase outcome prediction, and it works better than the measurement during occlusion.

OGG1 Inhibition Reduces Acinar Cell Injury in a Mouse Model of Acute Pancreatitis.

Acute pancreatitis (AP) is a potentially life-threatening gastrointestinal disease with a complex pathology including oxidative stress. Oxidative stress triggers oxidative DNA lesions such as formation of 7,8-dihydro-8-oxo-2'-oxoguanine (8-oxoG) and also causes DNA strand breaks. DNA breaks can activate the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) which contributes to AP pathology. 8-oxoG is recognized by 8-oxoG glycosylase 1 (OGG1) resulting in the removal of 8-oxoG from DNA as an initial step of base excision repair. Since OGG1 also possesses a DNA nicking activity, OGG1 activation may also trigger PARP1 activation. In the present study we investigated the role played by OGG1 in AP. We found that the OGG1 inhibitor compound TH5487 reduced edema formation, inflammatory cell migration and necrosis in a cerulein-induced AP model in mice. Moreover, TH5487 caused 8-oxoG accumulation and reduced tissue poly(ADP-ribose) levels. Consistent with the indirect PARP inhibitory effect, TH5487 shifted necrotic cell death (LDH release and Sytox green uptake) towards apoptosis (caspase activity) in isolated pancreatic acinar cells. In the in vivo AP model, TH5487 treatment suppressed the expression of various cytokine and chemokine mRNAs such as those of TNF, IL-1ß, IL1ra, IL6, IL16, IL23, CSF, CCL2, CCL4, CCL12, IL10 and TREM as measured with a cytokine array and verified by RT-qPCR. As a potential mechanism underlying the transcriptional inhibitory effect of the OGG1 inhibitor we showed that while 8-oxoG accumulation in the DNA facilitates NF-κB binding to its consensus sequence, when OGG1 is inhibited, target site occupancy of NF-κB is impaired. In summary, OGG1 inhibition provides protection from tissue injury in AP and these effects are likely due to interference with the PARP1 and NF-κB activation pathways.

Tricetin Reduces Inflammation and Acinar Cell Injury in Cerulein-Induced Acute Pancreatitis: The Role of Oxidative Stress-Induced DNA Damage Signaling.

Acute pancreatitis (AP) poses a worldwide challenge due to the growing incidence and its potentially life-threatening course and complications. Specific targeted therapies are not available, prompting the identification of new pathways and novel therapeutic approaches. Flavonoids comprise several groups of biologically active compounds with wide-ranging effects. The flavone compound, tricetin (TCT), has not yet been investigated in detail but sporadic reports indicate diverse biological activities. In the current study, we evaluated the potential protective effects of TCT in AP. TCT (30 µM) protected isolated primary murine acinar cells from the cytotoxic effects of cerulein, a cholecystokinin analog peptide. The protective effects of TCT were observed in a general viability assay (calcein ester hydrolysis), in an apoptosis assay (caspase activity), and in necrosis assays (propidium iodide uptake and lactate dehydrogenase release). The effects of TCT were not related to its potential antioxidant effects, as TCT did not protect against H2O2-induced acinar cell death despite possessing radical scavenging activity. Cerulein-induced expression of IL1ß, IL6, and matrix metalloproteinase 2 and activation of nuclear factor-κB (NFκB) were reduced by 30 µM TCT. In vivo experiments confirmed the protective effect of TCT in a mouse model of cerulein-induced AP. TCT suppressed edema formation and apoptosis in the pancreas and reduced lipase and amylase levels in the serum. Moreover, TCT inhibited interleukin-1ß (IL1ß), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) expression in the pancreas and reduced the activation of the oxidative DNA damage sensor enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Our data indicate that TCT can be a potential treatment option for AP.

Poly(ADP-Ribose) Polymerase 1 Promotes Inflammation and Fibrosis in a Mouse Model of Chronic Pancreatitis.

Chronic pancreatitis (CP) is an inflammatory disease of the pancreas characterized by ductal obstructions, tissue fibrosis, atrophy and exocrine and endocrine pancreatic insufficiency. However, our understanding is very limited concerning the disease's progression from a single acute inflammation, via recurrent acute pancreatitis (AP) and early CP, to the late stage CP. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor enzyme activated mostly by oxidative DNA damage. As a co-activator of inflammatory transcription factors, PARP1 is a central mediator of the inflammatory response and it has also been implicated in acute pancreatitis. Here, we set out to investigate whether PARP1 contributed to the pathogenesis of CP. We found that the clinically used PARP inhibitor olaparib (OLA) had protective effects in a murine model of CP induced by multiple cerulein injections. OLA reduced pancreas atrophy and expression of the inflammatory mediators TNFα and interleukin-6 (IL-6), both in the pancreas and in the lungs. Moreover, there was significantly less fibrosis (Masson's trichrome staining) in the pancreatic sections of OLA-treated mice compared to the cerulein-only group. mRNA expression of the fibrosis markers TGFß, smooth muscle actin (SMA), and collagen-1 were markedly reduced by OLA. CP was also induced in PARP1 knockout (KO) mice and their wild-type (WT) counterparts. Inflammation and fibrosis markers showed lower expression in the KO compared to the WT mice. Moreover, reduced granulocyte infiltration (tissue myeloperoxidase activity) and a lower elevation of serum amylase and lipase activity could also be detected in the KO mice. Furthermore, primary acinar cells isolated from KO mice were also protected from cerulein-induced toxicity compared to WT cells. In summary, our data suggest that PARP inhibitors may be promising candidates for repurposing to treat not only acute but chronic pancreatitis as well.

High-content screening identifies inhibitors of oxidative stress-induced parthanatos: cytoprotective and anti-inflammatory effects of ciclopirox.

BACKGROUND AND PURPOSE: Excessive oxidative stress can induce PARP1-mediated programmed necrotic cell death, termed parthanatos. Inhibition of parthanatos may be therapeutically beneficial in a wide array of diseases associated with tissue injury and inflammation. Our goal was to identify novel molecules inhibiting parthanatos. EXPERIMENTAL APPROACH: A small library of 774 pharmacologically active compounds was screened in a Sytox Green uptake assay, which identified 20 hits that reduced hydrogen-peroxide-induced parthanatos with an efficiency comparable to the benchmark PARP inhibitor, PJ34. KEY RESULTS: Of these hits, two compounds, antifungal ciclopirox and dopamine receptor agonist apomorphine, inhibited PAR polymer synthesis. These two compounds prevented the binding of PARP1 to oxidatively damaged DNA but did not directly interfere with the interaction between DNA and PARP1. Both compounds inhibited mitochondrial superoxide and H2 O2 production and suppressed DNA breakage. Since H2 O2 -induced damage is dependent on Fe2+ -catalysed hydroxyl radical production (Fenton chemistry), we determined the iron chelation activity of the two test compounds and found that ciclopirox and, to a lesser extent, apomorphine act as iron chelators. We also show that the Fe2+ chelation and indirect PARP inhibitory effects of ciclopirox translate to anti-inflammatory actions as demonstrated in a mouse dermatitis model, where ciclopirox reduced ear swelling, inflammatory cell recruitment and poly(ADP-ribosyl)ation. CONCLUSION AND IMPLICATIONS: Our findings indicate that the antimycotic drug, ciclopirox, acts as an iron chelator and thus targets an early event in hydrogen-peroxide-induced parthanatos. Ciclopirox has the potential to be repurposed as a cytoprotective and anti-inflammatory agent.

Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness.

Muscle spasticity after nervous system injuries and painful low back spasm affect more than 10% of global population. Current medications are of limited efficacy and cause neurological and cardiovascular side effects because they target upstream regulators of muscle contraction. Direct myosin inhibition could provide optimal muscle relaxation; however, targeting skeletal myosin is particularly challenging because of its similarity to the cardiac isoform. We identified a key residue difference between these myosin isoforms, located in the communication center of the functional regions, which allowed us to design a selective inhibitor, MPH-220. Mutagenic analysis and the atomic structure of MPH-220-bound skeletal muscle myosin confirmed the mechanism of specificity. Targeting skeletal muscle myosin by MPH-220 enabled muscle relaxation, in human and model systems, without cardiovascular side effects and improved spastic gait disorders after brain injury in a disease model. MPH-220 provides a potential nervous-system-independent option to treat spasticity and muscle stiffness.

Direct myosin-2 inhibition enhances cerebral perfusion resulting in functional improvement after ischemic stroke.

Acute ischemic stroke treatment faces an unresolved obstacle as capillary reperfusion remains insufficient after thrombolysis and thrombectomy causing neuronal damage and poor prognosis. Hypoxia-induced capillary constriction is mediated by actomyosin contraction in precapillary smooth muscle cells (SMCs) therefore smooth muscle myosin-2 could be an ideal target with potentially high impact on reperfusion of capillaries. Methods: The myosin-2 inhibitor para-aminoblebbistatin (AmBleb) was tested on isolated human and rat arterioles to assess the effect of AmBleb on vasodilatation. Transient middle cerebral artery occlusion (MCAO) was performed on 38 male Wistar rats followed by local administration of AmBleb into the ischemic brain area. Development of brain edema and changes in cerebrovascular blood flow were assessed using MRI and SPECT. We also tested the neurological deficit scores and locomotor asymmetry of the animals for 3 weeks after the MCAO operation. Results: Our results demonstrate that AmBleb could achieve full relaxation of isolated cerebral arterioles. In living animals AmBleb recovered cerebral blood flow in 32 out of the 65 affected functional brain areas in MCAO operated rats, whereas only 8 out of the 67 affected areas were recovered in the control animals. Animals treated with AmBleb also showed significantly improved general and focal deficit scores in neurological functional tests and showed significantly ameliorated locomotor asymmetry. Conclusion: Direct inhibition of smooth muscle myosin by AmBleb in pre-capillary SMCs significantly contribute to the improvement of cerebral blood reperfusion and brain functions suggesting that smooth muscle myosin inhibition may have promising potential in stroke therapies as a follow-up treatment of physical or chemical removal of the occluding thrombus.

Spilanthol Inhibits Inflammatory Transcription Factors and iNOS Expression in Macrophages and Exerts Anti-inflammatory Effects in Dermatitis and Pancreatitis.

Activated macrophages upregulate inducible nitric oxide synthase (iNOS) leading to the profuse production of nitric oxide (NO) and, eventually, tissue damage. Using macrophage NO production as a biochemical marker of inflammation, we tested different parts (flower, leaf, and stem) of the medicinal plant, Spilanthes acmella. We found that extracts prepared from all three parts, especially the flowers, suppressed NO production in RAW macrophages in response to interferon-γ and lipopolysaccharide. Follow up experiments with selected bioactive molecules from the plant (α-amyrin, ß-caryophylline, scopoletin, vanillic acid, trans-ferulic acid, and spilanthol) indicated that the N-alkamide, spilanthol, is responsible for the NO-suppressive effects and provides protection from NO-dependent cell death. Spilanthol reduced the expression of iNOS mRNA and protein and, as a possible underlying mechanism, inhibited the activation of several transcription factors (NFκB, ATF4, FOXO1, IRF1, ETS, and AP1) and sensitized cells to downregulation of Smad (TF array experiments). The iNOS inhibitory effect translated into an anti-inflammatory effect, as demonstrated in a phorbol 12-myristate 13-acetate-induced dermatitis and, to a smaller extent, in cerulein-induced pancreatitis. In summary, we demonstrate that spilanthol inhibits iNOS expression, NO production and suppresses inflammatory TFs. These events likely contribute to the observed anti-inflammatory actions of spilanthol in dermatitis and pancreatitis.